Evidence for the Selective Population of FeMo Cofactor Sites in MoFe Protein and Its Molecular Recognition by the Fe Protein in Transition State Complex Analogues of Nitrogenase

Grossmann, J. Günter; Hasnain, S. Samar; Yousafzai, Faridoon K.; Eady, Robert R.

doi:10.1074/jbc.m005350200

Cited by 10 publications

(9 citation statements)

References 46 publications

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“…For the MgADP-bound state of the native Fe protein, the structure has been crystallographically determined and the overall structure at a low resolution approximately resembles the conformation observed for the native Fe protein and in essence serves as an internal control for this analysis. Previous SAXS experiments probing the nucleotide-bound states of the Fe protein reported R g values for the nucleotide-bound conformations very similar to those observed for the nucleotide-free native Fe protein ( , ). In the current study, the effects of binding of nucleotide to both the native and L127Δ Fe protein are examined in parallel to directly assess whether the L127Δ Fe protein is a mimic of the MgATP state as suggested previously ().…”

Section: Resultssupporting

confidence: 68%

Probing the MgATP-Bound Conformation of the Nitrogenase Fe Protein by Solution Small-Angle X-ray Scattering

et al. 2007

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The MgATP-bound conformation of the Fe protein of nitrogenase from Azotobacter vinelandii has been examined in solution by small angle x-ray scattering (SAXS) and compared to existing crystallographically characterized Fe protein conformations. The results of the analysis of the crystal structure of an Fe protein variant with a Switch II single amino acid deletion recently suggested that the MgATP-bound state of the Fe protein may exist in a conformation that involves a large-scale reorientation of the dimer subunits resulting in an overall elongated structure relative to the more globular structure of the MgADP-bound state. It was hypothesized that the Fe protein variant may be a conformational mimic of the MgATP-bound state of the native Fe protein based largely on the observation that the spectroscopic properties of the [4Fe-4S] cluster of the variant mimicked in part the spectroscopic signatures of the native nitrogenase Fe protein in the MgATPbound state. In the present work, SAXS studies reveal that the large scale conformational differences between the native Fe protein and the variant observed by x-ray crystallography are also observed in solution. In addition, comparison of the SAXS curves of the Fe protein nucleotide-bound states to the nucleotide-free states indicates that the conformation of the MgATP-bound state in solution does not resemble the structure of the variant as initially proposed, but rather, at the resolution of this experiment, it resembles the structure of the nucleotide-free state. These results provide insights into the Fe protein conformations that define the role of MgATP in nitrogenase catalysis.

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Section: Resultssupporting

confidence: 68%

Probing the MgATP-Bound Conformation of the Nitrogenase Fe Protein by Solution Small-Angle X-ray Scattering

et al. 2007

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“…An A. vinelandii strain expressing the E146D Fe protein variant, which is specifically defective in FeMoco insertion, accumulates a homogeneous MoFe protein species (designated the E146D nifH MoFe protein), which has one P cluster and one FeMoco in one-half of the molecule and one P cluster and one vacant FeMoco site in the other half (36,50). Purifications of the wild-type MoFe protein from Klebsiella pneumoniae usually yield a portion of MoFe protein with the same cluster content as the E146D nifH MoFe protein in addition to the fully assembled MoFe protein species (58). This effect has been attributed to the instability of the wild-type MoFe protein from K. pneumoniae and could be further explained by the different stability of the two ␣␤ subunit pairs of this protein.…”

Section: Resultsmentioning

confidence: 99%

Characterization of Azotobacter vinelandii nifZ Deletion Strains

Fay

Santos

et al. 2004

Journal of Biological Chemistry

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The nifZ gene product (NifZ) of Azotobacter vinelandii has been implicated in MoFe protein maturation. However, its exact function in this process remains largely unknown. Here, we report a detailed biochemical/biophysical characterization of His-tagged MoFe proteins purified from A. vinelandii nifZ and nifZ/nifB deletion strains DJ1182 and YM6A (⌬nifZ and ⌬nifZ⌬nifB MoFe proteins, respectively). Our data from EPR, metal, activity, and stability analyses indicate that one ␣␤ subunit pair of the ⌬nifZ MoFe protein contains a P cluster ([8Fe-7S]) and an iron-molybdenum cofactor (FeMoco) ([Mo-7Fe-9S-X-homocitrate]), whereas the other contains a presumed P cluster precursor, possibly comprising a pair of [4Fe-4S]-like clusters, and a vacant FeMoco site. Likewise, the ⌬nifZ⌬nifB MoFe protein has the same composition as the ⌬nifZ MoFe protein except for the absence of FeMoco, an effect caused by the deletion of the nifB gene. These results suggest that the MoFe protein is likely assembled stepwise, i.e. one ␣␤ subunit pair of the tetrameric MoFe protein is assembled prior to the other, and that NifZ might act as a chaperone in the assembly of the second ␣␤ subunit pair by facilitating a conformational rearrangement that is required for the formation of the P cluster through the condensation of two [4Fe-4S]-like clusters. The possibility of NifZ exercising its effect through the Fe protein was ruled out because the Fe proteins from nifZ and nifZ/nifB deletion strains are not defective in their normal functions. However, the detailed mechanism of how NifZ carries out its exact function in MoFe protein maturation awaits further investigation.The biochemical machinery for the reduction of dinitrogen to ammonia is provided by the metalloenzyme nitrogenase. This enzyme comprises two separately purifiable proteins, the MoFe (molybdenum-iron) protein and the Fe (iron) protein (reviewed in Refs. 1-7 . This process is followed by complex dissociation, rereduction of the oxidized Fe protein, and dissociation of MgADP, allowing the enzyme complex to start the next cycle of electron transfer. Finally, electrons are believed to be transferred from the P cluster to FeMoco, where substrate binding and reduction take place.The assembly of nitrogenase and, in particular, the MoFe protein and its two unique metalloclusters is arguably one of the most complicated processes in the field of bioinorganic chemistry (reviewed in [15][16][17][18][19]and 21). Based on the current model, the assembly of the MoFe protein requires the participation of at least 15 different genes (9 -13, 15) and involves the following events: (i) the biosynthesis of FeMoco, (ii) the biosynthesis of a P cluster-containing yet FeMoco-deficient species of the MoFe protein in a separate pathway, and (iii) the insertion of completed FeMoco into the FeMoco-deficient MoFe protein (3, 9, 16 -19). It has been proposed that FeMoco biosynthesis starts with the mobilization of iron and sulfur and the assembly of Fe-S fragments that are subsequently transferred to NifB (nifB ge...

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“…It is possible to separate the MoFe protein of K. pneumonia into two species during purification by ion-exchange chromatography; one where both active sites are occupied by FeMoco and with an Mo content of 2.0 Mo · (MoFe protein) − 1 , and one where only one active site is occupied and containing 1.0 Mo · (MoFe protein) − 1 [15]. The specific activity of purified samples of the fully occupied MoFe protein was measured as 2300 nmol of C 2 H 2 reduced/min, whereas pure samples of the half-occupied MoFe protein have a specific activity of 1200 nmol of C 2 H 2 reduced/min.…”

Section: Stopped-flow Spectrophotometry Of Mofe Protein With Only Onementioning

confidence: 99%

Electron transfer and half-reactivity in nitrogenase

Clarke

Fairhurst

Lowe

et al. 2011

Biochemical Society Transactions

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Nitrogenase is a globally important enzyme that catalyses the reduction of atmospheric dinitrogen into ammonia and is thus an important part of the nitrogen cycle. The nitrogenase enzyme is composed of a catalytic molybdenum-iron protein (MoFe protein) and a protein containing an [Fe4-S4] cluster (Fe protein) that functions as a dedicated ATP-dependent reductase. The current understanding of electron transfer between these two proteins is based on stopped-flow spectrophotometry, which has allowed the rates of complex formation and electron transfer to be accurately determined. Surprisingly, a total of four Fe protein molecules are required to saturate one MoFe protein molecule, despite there being only two well-characterized Fe-protein-binding sites. This has led to the conclusion that the purified Fe protein is only half-active with respect to electron transfer to the MoFe protein. Studies on the electron transfer between both proteins using rapid-quench EPR confirmed that, during pre-steady-state electron transfer, the Fe protein only becomes half-oxidized. However, stopped-flow spectrophotometry on MoFe protein that had only one active site occupied was saturated by approximately three Fe protein equivalents. These results imply that the Fe protein has a second interaction during the initial stages of mixing that is not involved in electron transfer.

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Evidence for the Selective Population of FeMo Cofactor Sites in MoFe Protein and Its Molecular Recognition by the Fe Protein in Transition State Complex Analogues of Nitrogenase

Cited by 10 publications

References 46 publications

Probing the MgATP-Bound Conformation of the Nitrogenase Fe Protein by Solution Small-Angle X-ray Scattering

Probing the MgATP-Bound Conformation of the Nitrogenase Fe Protein by Solution Small-Angle X-ray Scattering

Characterization of Azotobacter vinelandii nifZ Deletion Strains

Electron transfer and half-reactivity in nitrogenase

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